ANTIMICROBIAL AND ANALGESIC ACTIVITIES OF TRIKATU CHURNA AND ITS INGREDIENTS
B. Uma Reddy* and Y.N. Seetharam
Department of Botany, Gulbarga University, Gulbarga Karnataka, India, Pin code -585 106
E-mail: drumareddy11@yahoo.co.in
Summary
Trikatu churna is one of the Ayurvedic herbal preparations, practice against certain general health disorders such as cold, cough, appetizer, carminative, purgative etc. In the present study phytochemical, antibacterial, antifungal and analgesic activities of ethanolic extracts of Trikatu churna (equiproportions of powdered fruits of Piper nigrum L,
Piper longum L and rhizome Zingiber officinale Roscoe.) and its
individual components were tested for antimicrobial activity against certain clinical bacterial and fungal isolates, such as Escherichia coli,
Staphylococcus aureus, Aspergillus niger and Mucor species by in-vitro
agar well diffusion method and analgesic activity by in-vivo hot plate method using mice. Trikatu churnawas found to possess higher the rate of phytoconstituents, promising antimicrobial (non-specific) and moderate analgesic activities.
Key Words: Trikatu churna; Agar well diffusion technique; Hot plate method; Antibacterial; Antifungal; Analgesic activity
Introduction
Trikatu churna is one of the traditional polyherbal preparation, formed by the mixture of three important spicy materials, such as Piper nigrum L. (Piperaceae), Piper longum L. (Piperaceae) and Zingiber officinale Roscoe. (Zingiberaceae) (1). All these plant materials are extensively used as one of the chief constituents in various Ayurvedic compound preparations (2) and healthcare preparations (3). The antioxidant, antitumor, anticancer and other pharmacological effects of P. nigrum, P. longum and Z. officinale
Few research studies have been carriedout on these individual ingredients, but so far no clinical trials have been made on this compound polyherbal formulation, Trikatu churna. Hence, the present study was undertaken for the systematic evaluation of the antimicrobial and analgesic activities of this polyherbal formulation, Trikatu churna and compared its effect to its individual ingredients.
Materials and Methods
The present study was undertaken to assess the antibacterial and antifungal activities of different concentrations of ethanolic extracts of Trikatu churna and its plant ingredients on highly pathogenic bacteria like Escherichia coli, Staphylococcus aureus few pathogenic fungi such as Aspergillus niger and Mucor species. Analgesic activity was studied the maximum pain tolerance of Trikatu churna in pain induced mice.
Collection of Plant materials: The plant materials of Piper nigrum L, Piper longum L along with the fruits were collected from the Agricultural University, Bangalore, rhizomes of Zingiber officinale Roscoe. were collected from the farmers of Gulbarga in the month of October-November. All these plants were authenticated and the voucher specimens are deposited in Herbarium of Department of Botany, Gulbarga University, and Gulbarga. The fruits and the rhizomes of respective plants were surface sterilized by using 50% alcohol, then shade dried and powdered for the preparation of Trikatu churna.
Preparation of Trikatu churna: The pre-cleaned dried powdered fruits of Piper
longum, P.nigrum and rhizomes of Zingiber officinale were taken in equal proportions
and mixed well. This churna was stored in an airtight container for further processing
Preparation of the Extracts: The 100g of Trikatu churna and its plant ingredients were extracted with 90% alcohol at 50 - 60°C in a soxhlet apparatus. The different extracts were collected in a separate container and concentrated to dryness in a flash evaporator (Buchi type) under reduced pressure and controlled temperature (40 - 50°C) and note down the yield of crude extracts.
Microorganisms Used: Clinical laboratory bacterial isolates of Staphylococcus aureus,
and Escherichia coli and pure fungal isolates like Aspergillus niger and Mucor species
obtained from the Department of Microbiology, Gulbarga University, Gulbarga.
The Preliminary Phytochemical Studies: The alcoholic extracts of Trikatu churna and its plant ingredients were tested by applying general chemical tests for alkaloids, tannins, steroids, terpenoids, phenols, flavonoids, saponins, etc (9-13).
Antimicrobial activity: Here the in-vitro antibacterial and antifungal activities were assayed by using agar well diffusion method. The pure cultures of different pathogens were grown overnight in sterile nutrient broth and incubated at 37°C for 24 hours, then subjected for optical density of the overnight incubated culture were adjusted to 0.1 at
λ600 with sterile nutrient broth. The 0.1ml of the culture was seeded on 25 ml of solidified nutrient agar plate and Sabouraud’s dextrose agar plates for bacterial and fungal cultures. The wells were bored with 8mm borer in seeded agar, and then the particular concentrations (500µg/0.2ml) of the extracts of Trikatu churna and its ingredients were added separately in each well. Soon after the plates were then kept at 10°C for 30min. After it normalized to room temperature plates were incubated at 37°C for 24hr. After incubation period was completed, the zone of inhibition was measured and recorded (14).
Analgesic activity: The analgesic activity of the ethanolic extract of Trikatu churna was studied by hot plate method (15). Swiss albino mice weighing between 20 -25g and heat sensitive were selected for experimental work. Then mice were randomly divided into five groups of 6 mice each. The first group served as control and received the vehicle only (ie., 0.2ml of distilled water per animal). The second, third, fourth, fifth and sixth groups of mice have received the alcoholic extract of Trikatu churna suspended in distilled water at 50, 100, 150, 200 and 250mg/kg b.wt., respectively. The seventh group received the standard analgin at a dose of 40mg/kg b.wt. All the treatments were administered intraperitoneally. The observations were made at 0, 30, 60 and 120 minutes. Statistical analysis: Results are expressed as mean + standard error of mean (SEM). The data obtained from the above studies were analyzed using Student’s paired‘t’ test. P values less than 0.05 were considered significant.
Results
The results demonstrated that, all the four extracts of Piper nigrum, Piper longum,
Zingiber officinale and Trikatu churna possess potent antimicrobial and moderate
analgesic activities.
Preliminary Phytochemical Screening: Qualitative Phytochemical analyses were carried out with the alcoholic extract of Trikatu churna and its ingredients in order to determine the presence of plant secondary metabolites. The results of the above studies clearly demonstrated the occurrence of alkaloids, phenols, tannins, flavonoids, steroids, lignins and saponins in all four different extracts. But the steroids are absent in Piper
nigrum, where as saponins and tannins are absent in Piper longum. Labat test for lignin is
Antibacterial activity: The clear circular zone of inhibition formed around the well containing the plant extract and the diameter of the inhibition zone was taken as the sensitivity index of the bacteria to plant extract. The data in the Table-1 indicates that, Trikatu churna was proved to be very effective against Escherichia coli and
Staphylococcus aureus, but the moderate activity was noticed with all the individual
ingredients of Trikatu churna against Escherichia coli and Staphylococcus aureus.
Table-1 Antibacterial activity of Trikatu churna and its ingredients. Zone of Inhibition (mm) Sl.
No Treatment
Concentration
(µg) Escherichia coli Staphylococcus
aureus
01 Piper nigrum L. 500 12 + 0.12 13 + 0.21
02 Piper longum L. 500 14 + 0.13 15 + 0.32
03 Zingiber officinale. Roscoe. 500 13 + 0.15 15 + 0.41
04 Trikatu churna 500 17 + 0.11 16 + 0.12 05 Streptomycin sulphate 50 20 + 0.12 19 + 0.01 06 Control (Sterile Distilled
Water
0.1 ml nil nil
Antifungal Activity: Inhibition of radial growth of the fungi over a period of 48 hours at 28°C in a medium amended with plant extract is taken as an indication of the antifungal activity of the plant extract as against the control. The data in the table-2 indicates that, the Trikatu churna and its ingredients have antifungal property. But the percentage inhibition of radial growth varies among them. Among, Piper nigrum has shown mild activity against both the fungi. Where as Piper longum and Zingiber officinale have shown significant antifungal activity, but Trikatu churna, a compound drug has shown highest activity against both the fungi tested
Table-2 Antifungal activity of Trikatu churna and its ingredients Zone of Inhibition (mm) Sl.
No Treatment
Concentration
(µg) Aspergillus niger Mucor species
01 Piper nigrum L. 500 4.1 + 0.10 3.9 + 0.01
02 Piper longum L. 500 5.5 + 0.14 5.0 + 0.12
03 Zingiber officinale. Roscoe. 500 5.8 + 0.11 5.5 + 0.11
04 Trikatu churna 500 6.2 + 0.01 7.0 + 0.05
05 Nystatin 50 9.0 + 0.12 10 + 0.11
06 Control (Sterile Distilled Water) 0.1 ml nil nil
Table-3. The analgesic activity of Trikatu churna on Swiss albino mice using hot platemethod
Pain Threshold (in seconds ) Sl.
No Treatment
Dose (mg/kg
b.wt)
15 min 30 min 45 min 60 min
01 Distilled Water 1.0ml 2.17 + 0.23 2.45 + 0.05 2.40 + 0.34 2.52 + 0.17 02 Trikatu churna 50 2.45 + 0.12 2.42 + 0.09 2.65 + 0.12 3.35 + 0.23 03 Trikatu churna 100 3.15 + 0.12 3.27 + 0.30 3.07 + 0.09 3.75 + 0.15 04 Trikatu churna 150 3.20 + 0.24 3.27 + 0.23 3.35 + 0.41 3.17 + 0.09 05 Trikatu churna 200 3.17 + 0.28 3.32 + 0.45 3.57 + 0.55 3.87 + 0.85 06 Trikatu churna 250 2.82 + 0.25 4.00 + 0.81 5.07 + 0.09 4.50 + 1.29 07 Analgin 40 4.25 + 0.95 7.71 + 0.91 9.42 + 0.50 8.70 + 0.14
Discussion
The increasing prevalence of multi drug resistant strains of bacteria and the recent appearance of strains with reduced susceptibility to antibiotics raises the specter of untreatable bacterial infections adds urgency to search for new infection fighting strategies. Several phytoconstituents like flavonoids and polyphenols (16), tannins (17), terpenoids (18), sesquiterpenes (19) have proven effective antimicrobial substances against a wide range of microorganisms.
The preliminary phytochemical observations of Trikatu churna and its individual ingredients have shown the occurrence of alkaloids, phenols, and essential oils, perhaps the combined effect of these groups of chemical compounds synergistically exhibited the inhibitory activity on the growth of bacteria and fungi. Since the analgesic activity is more connected to central nervous system, the active principles of the churna like piperine, gingerol, inoleresin and other active constituents of Piper nigrum, P. longum,
and Z. officinale might directly act upon the central nervous system. Further detailed
evaluation on the mechanism of action is needed. It is known that the commercially available analgesics causes irritability in the digestive system and also causes inflammation in allergic patients. Under such circumstances the Ayurvedic formulations such as churna which has carminative (20-21), and can also act as analgesic. This study would provide the preliminary scientific basis for the detailed phytochemical and pharmacological activity of such Ayurvedic preparations.
These above results also evidenced from the earlier reports on antimicrobial and analgesic activities of all the ingredients of Trikatu churna. The extract of Z. officinale
rhizome produced significantly inhibition of the carrageenan-induced rat paw oedema and a reduction in the number of writhing induced by acetic acid in mice (22-25). Antimicrobial and analgesic activities were explored from Piper species (26-27). The
Piper nigrum extract administered alone, showed significant decrease in writhes 78.43%
Conclusion
The extract of Trikatu churna was proved to be very effective against different bacterial and fungal isolates tested when compared to its individual ingredients. In fact these plants produce a wide range of bioactive molecules. Here, the combination of all these plant materials in equal proportions making the Trikatu churna preparation as one of the rich source of phytoconstituents and interaction of all these chemicals might be resulted in synergistically enhanced the therapeutic efficacy of antimicrobial and analgesic activities, thus lend pharmacological support to folkloric, ethnomedical and traditional uses of Trikatu churna for destruction of pathogenic microbes and increase the pain tolerance capacity in mice.
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